Use of hydroxyoleic acid and related compounds in the manufacture of drugs

ABSTRACT

Use of hydroxyoleic acid and its analogous compounds in the manufacture of drugs. Describes the use of hydroxyoleic acid and its analogs of general formula I: COOH—CHR—(CH 2 ) m —CH═CH—(CH 2 ) n —CH 3 , in which m and n have, independently, a value of 0-15 and R can be any residue with molecular weight below 200 Da, in the manufacture of drugs that can be used in the treatment of cancer, hypertension, obesity or diseases mediated by alteration of the membrane structure and the consequent regulation of G-proteins or of the receptors coupled to them.

This application is a divisional of application Ser. No. 10/488,726filed on Aug. 26, 2004 now abandoned which is a 371 of PCT/ES02/00475filed on Oct. 9, 2002, which designated the U.S., claims the benefitthereof and incorporates the same by reference.

FIELD OF THE INVENTION

The present invention relates to the use of hydroxyoleic(2-hydroxyoleic) acid and molecules of a similar structure as antitumoragents, as agents with hypotensive activity and as agents for inducingreductions in body weight.

The present invention also relates to the use of 2-hydroxyoleic acid andsimilar compounds for controlling membrane structure, controlling theactivity and/or localization of G-proteins and controlling the activityof receptors bound to G-proteins through regulation of membranestructure.

The present invention also relates to the use of 2-hydroxyoleic acid andsimilar compounds for the manufacture of drugs intended for cancertreatment, of drugs for treating cardiovascular diseases and of drugsfor treating problems of body weight and obesity.

BACKGROUND TO THE INVENTION

Fatty acids are molecules of wide application, both in foodstuffs and inindustry. 2-Hydroxyoleic acid, the synthesis of which has been describedpreviously (Adam et al., 1998, Eur. J. Org. Chem. 9, 2.013-2018), hasbeen used industrially as an emulsifier for preparations of cosmetics.

For example, on the one hand, patent JP 10182338 relates to anoil-in-water emulsifying composition that exhibits low irritability andhigh compatibility with salts, which contains: [A] nonionic surfactantssuch as polyoxyethylene sorbitol monolaurate, polyoxyethylene sorbitolmonooleate and polyoxyethylene sorbitol monostearate, [B] 2-hydroxyC10-C22 fatty acids such as 2-hydroxystearic acid, [C] oils and [D]water, in which the A/B ratio is between 1:0.01 and 1:2.

Patent JP 09110635 also relates to compositions that can be used aspharmaceutical products, cosmetics and foodstuffs and contain: [A]esters of polyglyceryl fatty acid, [B] 2-hydroxy C10-C22 fatty acids,[C] oils and [D] water, where the weight ratio of A/C and B/C is 2.0 and0.5 respectively, and have average particle sizes between 10 and 300 nm.These compositions show good stability even in acid conditions or at lowviscosity or in the presence of elevated quantities of salts, andtherefore are compatible with the skin.

On the other hand, this fatty acid has also been employed as aninhibitor of oleamide hydrolase, an action that is associated with thesleep inducing effect of this substance (patents U.S. Pat. No. 6,096,784and WO 9749667).

For example, patent U.S. Pat. No. 6,096,784 relates to the design andsynthesis of oleamide hydrolase inhibitors, responsible for thehydrolysis of a sleep-inducing lipid (cis-9-octadecenamide). The mostpotent inhibitors possess an electrophilic carbonyl group capable offorming, reversibly, a (thio)hemiacetal or a (thio)hemiacetal forimitating the transition state of a reaction catalyzed by a protease ofthe cisteine or serine type. In addition to the inhibitory activity,some of the inhibitors displayed agonistic activity that induces sleepin laboratory animals.

The Hexagonal Membrane Structures.

The membrane lipids are able to arrange themselves in a greater numberof secondary structures than the proteins and nucleic acids. The typicallipid bilayer of biological membranes is just one of these secondaryconfigurations. Little is known about the abundance and roles of othersecondary structures in living cells. One function of these structureswas described in a previous work of the inventors: to increase thebinding affinity of G-proteins to membranes (Escribá PV, Ozaita A, RibasC, Miralles A, Fodor E, Farkas and Garcia-Sevilla J A; 1997 Proceedingsof the National Academy of Sciences of the USA 94, 11375-11380).

The concept of membrane structure goes far beyond that described in someof the patents of the state of the art (WO 87/04926 and WO 80/11286), inwhich only membrane fluidity is mentioned, and the concept is extendedto a much wider field: the membrane structure. The molecules covered byour patent act on the transition or passage from lamellar to hexagonalstructure (FIG. 1).

Examining the prior art cited, in the state of the art there are noother applications connected with 2-hydroxyoleic acid or similarcompounds that would be of particular interest in the area of cancertreatment, cardiovascular diseases and/or control of body weight.

There are only descriptions of dietary products (GB 2140668, EP 0611568and WO 02/0042) or extracts from cultures of M. cryophilus (WO89/11286), which consist of complex mixtures of various compounds thatinclude some of those described in the invention, such as fatty acids,in particular oleic and palmioleic acids, for example, and the use ofthese mixtures in the treatment of arterial hypertension, for thecontrol of obesity or as antitumor agents, but without ascribing to anyof the components of the mixture considered in the present invention, aspecific role in the said therapeutic effect. Only WO 02/051406 and WO94/01100 describe the use of certain fatty acids (C₁₄-C₂₀) in thetreatment of prostate cancer, which is not an object of the presentinvention, exclusively when using the said fatty acids described in thestate of the art.

AIM OF THE INVENTION

The present invention has the aim of finding new applications of2-hydroxyoleic acid and similar compounds that are unconnected withthose described in the state of the art.

A first objective of the present invention is to show that2-hydroxyoleic acid and its analogs possess activity as antitumoragents.

A second objective of the present invention is to show that2-hydroxyoleic acid and its analogs possess activity as hypotensiveagents.

A third objective of the present invention is to show that2-hydroxyoleic acid and its analogs possess activity as agents thatinduce a reduction in body Weight.

A fourth objective of the present invention is to show that2-hydroxyoleic acid and its analogs have application as agents forcontrolling the transition from lamellar to hexagonal structure of cellmembranes. This regulation of membrane structure has an influence on theactivity of G-proteins, as well as of the molecular entities of theirtransduction pathway, i.e. of their route of signal propagation. A largenumber of drugs acts on the receptors bound to G-proteins by directinteraction with molecules of this type or with the mechanisms connectedwith the cell signals derived from their activity. 2-Hydroxyoleic acidand its analogs, however, act upon the membrane structure.

The applications described below for 2-hydroxyoleic acid and its analogshave not been cited by anyone previously and their use may provebeneficial for the treatment of certain pathologies. In particular, ithas been found that 2-hydroxyoleic acid and its analogs displayantitumor activity, hypotensive (or antihypertensive) activity andinduce reduction of body weight.

In the present invention the new applications of 2-hydroxyoleic acid andits analogs are substantiated by using experimental models, inparticular systems of in vitro analysis, cell culture systems and livingorganisms. All these analysis models show, without doubt, that2-hydroxyoleic acid and its analogs are molecules that can be used formaking drugs for cancer treatment, for treatment of cardiovasculardiseases and for treating subjects with problems of body weight andobesity, as well as other diseases or deficiency conditions based on thecontrol of signals associated with G-proteins, mediated by the lamellarto hexagonal transition of the membrane structure.

DESCRIPTION OF THE INVENTION

In the present invention, “2-hydroxyoleic acid” means α-hydroxyoleicacid, octadecenoic acid C18:1 cis Δ9 or cis-2-hydroxy-9-octadecenoicacid. “Analogs” means those fatty acids that have the double bondshifted one or two positions from the central zone and/or that have thedouble bond shifted from one to five positions from the central zoneand/or have from one to six carbon atoms (CH₂ groups) more or less oneach side of the double bond and/or that have a residue (R) in position2 different from OH, with a small atomic mass (Mw less than or equal to200 Da). It does not matter whether the stereoisomer corresponding tothe projection of the R group is R or S. In relation to the variousmolecules tested, it has been observed that those having the generalformulas shown below display some similar effects to hydroxyoleic acidand can therefore be categorized as analogs thereof.

General formula I: COOH—CHR— (CH₂)_(n)—CH═CH— (CH₂)_(n)—CH₃ in which mand n have, independently, a value of 0-15 and R can be H, OH, NH₂, CH₃,or some other residue with molecular weight below 200 Da.

In the present invention “G-proteins” means proteins that are guaninenucleotide binding proteins, formed from three subunits (one alpha, onebeta and one gamma) that transmit signals from receptors bound toG-proteins, to effectors (adenylyl cyclase, guanylyl cyclase,phospholipase C, ion channels, etc.).

In the present invention “membrane structure” means the secondarystructure or arrangement of lipids in natural or synthetic membranes(liposomes).

In the present invention “acute effect” means the effect that isproduced in a space of time between minutes and some hours after asingle administration of a drug.

In the present invention “chronic effect” means the effect that isproduced in a space of time between a few days and several weeks ofcontinuous administration of a drug.

In the present invention “pharmaceutically acceptable forms” means anyof those used routinely in the sector, including, non-limitatively:esters, especially ethyl esters for their properties as solubilizers offatty acids, ethers, amides, salts, etc.

-   -   One objective of the present invention is the application of        2-hydroxyoleic acid and its analogs in controlling the        transition from lamellar to hexagonal structure of the cell        membranes. The molecular basis of this phenomenon lies in the        interaction of 2-hydroxyoleic acid and its analogs with        membranes and in modulation of the membrane composition and/or        structure (Tables 1 and 2).

TABLE 1 Effect of binding of 2-hydroxyoleic acid on the temperature oftransition from lamellar to hexagonal structure (H_(II)). DEPE:2OHOA(mol:mol) T_(H) ^(a) (° C.)  1:0 (pure DEPE) 63 40:1 54 20:1 48 10:1 41^(a) T_(H) indicates the temperature of transition from lamellar tohexagonal structure.

Table 1 shows the values of the temperature of transition from lamellarto hexagonal structures in membranes of dielaidoylphosphatidylethanolamine (DEPE). The control value (in the absence of2-hydroxyoleic) is 63° C. The decrease (concentration-dependent) inducedby 2-hydroxyoleic acid (20HOA) shows that this molecule stabilizes thepresence of non-lamellar structures. This important modification of thecell membrane has important consequences for molecular and cellularfunction. All the analogs of 2-hydroxyoleic tested that have therapeuticactivity also induce effects on the membrane structure (Table 2).

TABLE 2 Effect of various analogs of hydroxyoleic acid(phospholipid:analog 20:1 mol:mol) on the lamellar-hexagonal transitionof the phospholipid dielaidoyl phosphatidylethanolamine (DEPE) T_(H) (°C.) Control (DEPE only) 61 Oleic acid 45 Aminoleic acid 49 Methyloleicacid 50 cis-Vaccenic acid 53 Nervonic acid 55Modulation of the Lamellar to Non-Lamellar Transition Regulates theActivity of G-Proteins.

Hydroxyoleic acid and related compounds are capable of modulating theactivity of G-proteins, measured by the binding of [³⁵S] GTPγS (FIG. 5).

This is because these molecules have an influence on the interaction ofG-proteins with membranes and therefore on their cellular localization,as is shown in the photographs from confocal microscopy (FIG. 2). Theeffect of hydroxyoleic acid and its analogs on the localization andactivity of G-proteins is not due to a direct interaction on them. FIG.6 shows the effect that these molecules have on the activity of purifiedG-proteins, in the absence of membrane. In contrast to what occurs whenthe G-proteins are bound to membranes, daunomycin (DNM) and hydroxyoleic(2OHOA), as well as the analogs of the latter, did not have an influenceon the activity of these proteins (which are activated only when theyare in membranes in contact with receptors bound to G-proteins).

Receptors bound to G-proteins are ubiquitous, making up 80% of themembrane receptors that transmit signals initiated by neurotransmitters,hormones, neuromodulators, cytokines, growth factors, etc. Among otherphysiological processes, they regulate blood pressure, cell growth andproliferation, and body weight. Accordingly, the molecules described inthis invention can regulate the aforementioned physiological processes.

-Tests on Membrane Structure-

The most effective and powerful technique for investigating membranestructure is X-ray diffraction/scattering. Using this technique, weestablished that the structure of the membrane is altered by2-hydroxyoleic acid and its analogs. The lowering of the temperature oftransition from lamellar to hexagonal structure indicates an importanteffect on rearrangement of the lipid molecules in the membrane. Thisregulation of rearrangement forms the basis of the effect exerted by2-hydroxyoleic acid and its analogs. All the analogs studied, whichcomply with the general formula, possess activity of membrane modulationand control of cell proliferation (efficacy in cancer), of bloodpressure (efficacy in cardiovascular processes) and of body weight(efficacy in obesity).

-   -   An objective of the present invention is to demonstrate that        2-hydroxyoleic acid and its analogs possess activity as        antitumor agents.

Firstly, the cell cycle is controlled by growth factors which bind tospecific receptors on the cell surface. Binding of the said growthfactors to the receptors gives rise to a cascade of reactions that areintended to activate mitogenic kinases (cdk) that form dimeric complexeswith the proteins associated with the cell cycle called cyclins. Thecdk/cyclin complexes regulate the phases of the cell cycle and itsprogression to produce mitosis and cell division.

Many specific receptors on the cell surface are bound to G-proteins, sothat when the growth factor interacts with the receptor the G-protein isactivated, triggering the cascade of reactions mentioned earlier.

Accordingly, modulation of the localization and activity of G-proteinswill make it possible to control cell growth and cell division.

The mechanism associated with the antitumor effects of 2-hydroxyoleicacid and its analogs is based on the fact that they induce modulation ofthe localization and activity of G-proteins and other peripheralproteins, such as protein kinase C or the small G-proteins (of the typeRas, Raf, etc.). This modulation is associated with regulation of thestructure of the membrane lipids.

It has been found that 2-hydroxyoleic acid acts as an inhibitor oftranslocation of G-proteins to the nucleus (FIG. 2). In this way,inhibition of cell proliferation is achieved, as was confirmed by theappreciable and significant increases in the levels of the protein p21and the decreases in the cell cycle proteins cdk2 and cyclins B and D3(FIGS. 3 A and B). Moreover, it has been observed that in cells inculture, 2-hydroxyoleic acid and its analogs induce significantincreases in protein kinase C (increase of between 40% and 120%).

This change in cellular localization of G-proteins produces modulationsin their function, greater than those produced by the drug daunomycin,widely used in the treatment of cancer. These changes have importantinhibitory effects on the proliferation and survival of tumor cells(FIG. 4).

An important regulator of the cell cycle is the protein p53, whichexerts a negative type of control by slowing down cell division at thelevel of G1 (the stage before mitosis). This protein is synthesized bythe cell itself in response to the appearance of alterations of the DNA.If the replicated DNA can have a negative influence on the daughtercells, the p53 protein is activated, giving rise to apoptosis(programmed cell death). Activation of the said protein p53 means thatother genes are expressed that code for regulator proteins such as p21,p27, p16, etc., which inhibit the activity/expression of the cyclins andcdks (involved in the process of the cell cycle).

In many types of tumor cells, the p53 protein appears to be mutatedand/or inactive, and proliferation of transformed (cancerous) cellsoccurs. The presence of 2-hydroxyoleic acid and/or its analogs in thecells induces activation of the signal pathway associated with p53,which induces the start of apoptosis or stopping of the cell cycle invarious types of tumor cells. With the aim of carrying out the firstobjective of the present invention, in vitro and in vivo models wereused. In this connection, 2-hydroxyoleic acid and all the structuralanalogs that comply with general formula I described earlier have beenshown to possess considerable antitumor capacity. The molecules that areanalogs of 2-hydroxyoleic (2-hydroxy-cis-9-octadecenoic) acid testedwere: 2-methyl-oleic (2-methyl-cis-9-octadecenoic) acid, 2-amino-oleic(2-amino-cis9-octadecenoic) acid, oleic (cis-9-octadecenoic) acid,palmitoleic (cis-9-hexadecenoic) acid, cis-vaccenic(cis-11-octadecenoic) acid and nervonic (cis-15-tetracosenoic) acid.These molecules have been shown to halt cell proliferation or induce thedeath of various types of tumor cells (e.g. human lung cancer cellsA549, Jurkat T lymphocytes, etc.) (FIG. 4). These results demonstratethe antitumor activity of the fatty acids described and show that thebasic structure of 2-hydroxyoleic acid can have small variations withoutaltering its antitumor activity.

Other molecular models enabled us to confirm that 2-hydroxyoleic acidand its analogs possess antitumor activity that is greater than thatpresented by other antitumor drugs, for example the anthracyclines, andthey are therefore compounds of high therapeutic interest. In membranesof 3T3 fibroblasts, we succeeded in proving that the presence of 200 μMof 2-hydroxyoleic acid and/or oleic acid (one of the analogs of2-hydroxyoleic acid) induces inhibition of 75-84% in the activity ofG-proteins in NIH 3T3 cells, whereas 200 μM of daunomycin induces aninhibition of 46% in the said activity (FIG. 5).

The antitumor efficacy of 2-hydroxyoleic acid and its analog oleic acidis shown in FIG. 8, in which we can see the disappearance of somecerebral tumor metastases, formed from a lung adenocarcinoma, aftertreatment with 2-hydroxyoleic acid. Treatment with this 2-hydroxyoleicacid and oleic acid caused complete disappearance of the cancer. Theseresults demonstrate that (a) 2-hydroxyoleic acid and its analogs aremolecules that can be used for making drugs intended for treatingcancer; (b) that they have a broad spectrum of action (they have beeneffective on various types of tumor cells in culture and in livingorganisms) and (c) that they are superior to other molecules used forthe treatment of cancer in their antitumor potency, in their absence ofside effects and in that they are administered orally, althoughintravenous or subcutaneous administration is also possible.

-Antitumor Tests-

The antiproliferative efficacy of hydroxyoleic acid has beendemonstrated in human lung cancer cells A549 and in human leukemia cells(Jurkat). FIG. 3 shows the induction of the antiproliferative proteinp21, which is accompanied by a decrease in proteins cdk2, cyclin B andcyclin D3, necessary for the tumor cells to be able to divide. Similareffects were produced by all the analogs tested that comply with thegeneral structural formula given earlier. This antiproliferative effectof 2-hydroxyoleic acid and its analogs was demonstrated by the reductionin cell density in tumor cells in culture (FIG. 4). Thisantiproliferative effect was also observed using other techniques andother cell types: in rat primary astrocytes, these fatty acids have anantiproliferative effect, which I studied by incorporating tritiatedthymidine. Moreover, hydroxyoleic acid and its analogs are able toinduce apoptosis or programmed cell death in human cancer cells. On theone hand, the degradation of PARP (FIG. 3C), and on the other hand thechange in cellular morphology and the presence of cell residues (FIG. 4)demonstrate the effect of 2-hydroxyoleic acid and its analogs asinducers of cell death. Using flow cytometry experiments, it wasestablished that in the presence of 2-hydroxyoleic acid the number oflive human leukemia cells (Jurkat) was only 10% of those that remainedalive with Etoposide, a known antitumor agent.

Finally, investigation of the effect in humans has revealed that2-hydroxyoleic acid and its analogs may constitute a family of antitumordrugs of great importance. FIG. 8 shows the effect of treatment with2-hydroxyoleic acid and oleic acid on tumors in humans. The case showncorresponds to a female patient in whom previous chemotherapy andradiotherapy did not produce reductions of brain tumors.

-   -   An objective of the present invention is to demonstrate that        2-hydroxyoleic acid and its analogs are agents with hypotensive        activity.        Regulation of the Activity of G-Proteins Controls Blood        Pressure.

There is a relationship between the drugs of the invention and theactivity of G-proteins and blood pressure. A result that confirms whatwas described earlier is a study carried out in humans, where it wasseen that hypertensive individuals have changes in the levels ofmembrane lipids (Table 3). The membrane lipids have an influence on thelamellar-hexagonal transition, which in its turn determines thelocalization and functionality of G-proteins. In fact, in patients withhypertension, we observe a change in the levels of G-proteins bound tothe membrane which is due to the aforementioned change in membranelipids and the ease of forming hexagonal phases. If the modulation ofnon-lamellar membrane structures and the consequent relocalization ofthe G-proteins produces hypertension, by regulating thelamellar-hexagonal transition of the membrane lipids it is possible toachieve regulation of the localization of the membrane proteins and,finally, of blood pressure (FIG. 7).

TABLE 3 Composition of fatty acids of phospholipids and esters ofcholesterol in erythrocyte membranes in normotensive (control) andhypertensive subjects (mg/100 mg). Phospholipids Esters PatientsPatients with with Cholesterol Fatty acid hypertension Controlshypertension Controls C14:0 0.4 ± 0.1 0.2 ± 0.1 0.9 ± 0.3 1.0 ± 0.2 C14:1n-5 1.7 ± 0.2  2.2 ± 0.3** 0.7 ± 0.2 n.d.*** C16:0 23.7 ± 0.6  23.1± 0.5  5.1 ± 0.6 14.3 ± 0.5*  C16:1n-9 0.4 ± 0.0 0.3 ± 0.0 3.1 ± 0.6 2.5± 0.3** C16:1n-7 0.5 ± 0.0 0.5 ± 0.1 2.4 ± 0.4  1.7 ± 0.1*** C16:4n-32.6 ± 0.3 2.5 ± 0.5 n.d. n.d. C18:0 16.6 ± 0.3  17.1 ± 0.6  3.2 ± 0.52.9 ± 0.5  C18:1n-9t 0.9 ± 0.1   0.6 ± 0.0*** n.d. n.d. C18:1n-9 16.3 ±0.6  16.0 ± 0.8  18.4 ± 1.2  16.7 ± 0.6**  C18:1n-7 1.8 ± 0.1  2.0 ±0.2** 1.5 ± 0.1 1.7 ± 0.2** C18:2n-6 12.5 ± 0.7  13.4 ± 0.7* 45.8 ± 2.8 51.1 ± 1.6*** C18:3n-6 0.4 ± 0.0 n.d.*** 0.9 ± 0.2 0.8 ± 0.1** C18:3n-30.3 ± 0.0 0.4 ± 0.1 n.d. n.d. C20:2n-6 2.1 ± 0.1 2.1 ± 0.2 0.9 ± 0.1 0.9± 0.1  C20:4n-6 17.0 ± 0.4  16.5 ± 0.4  7.0 ± 0.6 6.4 ± 0.9*  C22:4n-60.7 ± 0.3 0.6 ± 0.1 n.d. n.d. C22:6n-3 0.7 ± 0.1 0.8 ± 0.2 n.d. n.d.C24:1n-9 1.5 ± 0.1 1.6 ± 0.1 n.d. n.d. Total SFA 41.2 ± 1.1  41.1 ± 0.7 19.3 ± 1.2  18.2 ± 1.1*  Total MUFA 22.6 ± 0.6  21.7 ± 0.7* 25.9 ± 1.6 22.7 ± 0.8*** Total PUFA 36.2 ± 1.2   38.2 ± 0.9** 54.8 ± 2.4  59.1 ±1.6*** PUFA:SFA  0.8 ± 0.04  0.9 ± 0.03 2.7 ± 0.3  3.4 ± 0.3***PUFA:MUFA 1.6 ± 0.1 1.7 ± 0.1 2.2 ± 0.2  2.6 ± 0.2*** The values aremean values ± standard error of the mean (n = 28). SFA, saturated fattyacids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fattyacids. *P < 0.05, **P < 0.01, ***P < 0.001. n.d.: not detected.

It was demonstrated that 2-hydroxyoleic acid and its analogs also have amarked hypotensive effect, since they induce reductions in systolic anddiastolic blood pressure, without altering the heart rate (FIGS. 9 a, 9b and 10). The hypotensive effect produced by 2-hydroxyoleic acid andits analogs is that it induces a reduction in blood pressure of an acuteform (which is evident from 2 hours of treatment) and chronic (which ismaintained for days and weeks while the treatment is maintained).Aminoleic acid, for example, reduces blood pressure by 15 mmHg inchronic treatments lasting a week.

Blood pressure is controlled by various systems of receptors coupled toG-proteins, such as vasopressin receptors, adrenergic receptors, etc.

Interaction between the hormones involved in the control of bloodpressure with the receptors bound to stimulating G-proteins is modulatedby the action of 2-hydroxyoleic acid and similar molecules.

These fatty acids regulate communication between receptor, G-protein andeffector. The result is a modulation of the signals of cyclic AMP,phospholipase C and nitric oxide, which gives rise to a reduction inblood pressure. This effect is also connected with regulation of themembrane structure. The main pharmacological advantage of 2-hydroxyoleicacid and its analogs is that, in contrast to other hypotensive drugs,they do not have effects on the heart rate (i.e. they do notsignificantly increase or decrease the heart rate). An additionaladvantage of these compounds is that they control other cardiovascularrisk factors: the serum lipid/lipoprotein profile and body weight (seebelow). Since control of blood pressure is not sufficient on its own toprolong a patient's life and that it is necessary to control othercardiovascular risk factors, these fatty acids are superior to othermolecules employed in patients with cardiovascular diseases.

-Hypotensive tests-

2-Hydroxyoleic acid induced significant reductions of blood pressure inrats (FIG. 9). These reductions were of an acute form (at 2 hours oftreatment, 19±6 mmHg, P<0.01, n=6) and of a chronic form (1 week, 26±7mmHg, P<0.001, n=6). Furthermore, acute and chronic treatments from 1mg/kg to 10 mg/kg also produced significant reductions in blood pressurethat were concentration-dependent.

In humans, 2-hydroxyoleic acid also produced significant, largedecreases in blood pressure, see FIG. 9.

Moreover, the analogs of 2-hydroxyoleic acid that comply with thegeneral formula given above had a hypotensive effect (FIG. 10). In allcases, the decreases in blood pressure were significant in comparisonwith the controls (*P<0.05, **P<0.01).

These results clearly show that 2-hydroxyoleic acid and its analogs areeffective agents for clinical/pharmacological treatment of high bloodpressure.

-   -   An objective of the present invention is to demonstrate that        2-hydroxyoleic acid and its analogs possess activity as agents        that induce reductions in body weight (FIG. 11).

In addition to the antitumor and hypotensive activity of 2-hydroxyoleicacid and its analogs, they induce reductions in body weight.

Body weight is regulated by, among other things, factors such as theindividual's metabolic capacity and control of food intake.

Control of food intake is determined by the feeling of satiety, which inturn is regulated at the hormonal level. For example, deficiency ofnutrients stimulates the secretion of hormones which give rise to asensation of appetite. After eating, once the nutrient levels have beenrestored, there is stimulation of the secretion of hormones that giverise to a feeling of satiety.

It has been found that 2-hydroxyoleic acid and its analogs produceeffects of satiety, inducing reductions in food intake. This control isalso mediated by receptors of cytokines, leptins, adrenoceptors, andother receptors coupled to G-proteins, whose activity is modulated bythese fatty acids.

In the animals treated, this effect on satiety meant a consumption offeed between 15% and 30% less than the control animals.

-Tests of Control of Body Weight-

Rats treated with these molecules lost body weight during chronictreatments (from 5 to 17 days). In these experiments, rats treated with2-hydroxyoleic acid or its analogs, in particular aminoleic acid, hadfree access to food and water, in the same way as the control group oftreated rats (FIG. 11). In these conditions there was a progressivedecrease in the rats' body weight starting from the first day oftreatment, up to 17 grams on the seventh day of treatment (5% of thenormal body weight of a Sprague-Dowley rat aged 2-3 months). The feedsupplied to these animals was weighed and the consumption was found tobe lower during the treatment time, confirming that the treatments withthe molecules relating to this invention produce an effect of satiety inthe animal. Similar experiments carried out on adult mice, for periodsof up to 28 days with 2-hydroxyoleic acid, show reductions in bodyweight from 15% to 25%, relative to control mice (treated with vehicle).

DESCRIPTION OF THE DIAGRAMS

FIG. 1: Some of the many structures, in addition to lamellar, that themembranes can adopt.

FIG. 2 shows the cellular localization of Gαi₂ protein labeled withfluorescein in primary astrocytes of rat brain. In control cells, thelabeling indicates the presence of this protein throughout the cell,especially in the nucleus (arrow). In cells treated with 2-hydroxyoleic(2OHOA), the labeling appears in membrane and cytosol, but not in thenucleus (arrow).

FIG. 3 shows the effect of 2-hydroxyoleic acid in molecular markers ofcell proliferation and cell death. Part A shows the effect of2-hydroxyoleic acid (2OHOA) on the cell cycle proteins cdk2, cyclin Band cyclin D3 in human lung cancer cells A549. The decrease in theseproteins shows that this fatty acid induces stopping of the cell cycle,i.e. stopping of cell division. Part B shows the effect of2-hydroxyoleic acid (2OHOA) on p21 protein in A549 cells afterincubation for 24 and 48 hours. Protein p21 inhibits the cell cycle, soit is an antiproliferative protein. The large increases that2-hydroxyoleic acid induces on this protein explain the stopping of thecycle and the proliferation of tumor cells. Part C shows the degradationof poly-ADP ribose polymerase (PARP) in human leukemia cells (Jurkat)(Etoposide: 25 [E1] and 250 μM [E2]; 2-hydroxyoleic acid: 10[O1], 100[O2] and 1000 μM [O3]). The decrease in levels of this enzyme, orevidence of its degradation, indicate the start of apoptosis orprogrammed cell death. In these experiments Etoposide was used as thepositive control, as this molecule is known to be an inducer ofapoptosis and an antitumor agent.

FIG. 4 shows the effect of 2-hydroxyoleic acid and its analogs on theproliferation of human lung cancer cells A549 (A) and Jurkat cells ofhuman leukemia (B). Both 2-hydroxyoleic acid (2OHOA) and its analogs,all of which comply with the formula given previously, induce stoppageof division and the death of tumor cells (OA: oleic acid; VA:cis-vaccenic acid; POA: palmitoleic acid; NA: nervonic acid; 2MOA:2-methyl oleic acid; 2NOA: 2-amino oleic acid.

FIG. 5 shows the binding of [³⁵S] GTPγS to membranes of NIH 3T3 cellstransfected with the rat adrenoceptor α_(2A/D). This parameter measuresthe activity of G-proteins. The presence of 2-hydroxyoleic induces adecrease in function of the G-proteins even greater than theanthracycline daunomycin (DNM). The anthracyclines are potent antitumordrugs, therefore 2-hydroxyoleic is potentially more effective againsttumors. The analogs of 2-hydroxyoleic produce an effect similar to thatof 2-hydroxyoleic acid.

FIG. 6: Hydroxyoleic acid and its analogs had no effect on pureG-proteins (in the absence of membrane). This shows that its effect onthe activity of G-proteins is mediated by the regulation of thenon-lamellar membrane structures. Daunomycin (DNM) had a behaviorsimilar to the control.

FIG. 7: Levels of G-proteins in membranes of erythrocytes ofnormotensive subjects (empty bars) and hypertensive subjects (filledbars). The levels of proteins Gαi_(1/2)(Gi), Gαo (Go), Gαs (Gs) and G Gβ(Gb) are significantly lower in hypertensive subjects. The values of thebars are mean values standard error of the mean *P<0.05, **P<0.01.

FIG. 8 shows brain metastases (tumors) formed from a lungadenocarcinoma. The image on the left (8 a) corresponds to the tumorsbefore treatment and those on the right (8 b, 8 c and 8 d) correspond tothe tumors after treatment with 2-hydroxyoleic on various dates. As canbe seen, one of the tumors disappeared more quickly and the other onemore slowly.

FIG. 9 a shows the acute effect (2 hours, black bars) and chronic,effect (3 daily injections for 7 days, white bars) of hydroxyoleic acid(30 mg/kg) on the systolic arterial pressure in Sprague-Dowley rats.Lower doses of this molecule (1-10 mg/kg) produced similar effects, butless marked. *P<0.01.

FIG. 9 b shows the effect of 2-hydroxyoleic acid (30 mg/kg) on bloodpressure in humans. This diagram shows systolic arterial pressure as afunction of the day of treatment. The days prior to treatment are shownwith negative values. *P<0.05, **P<0.01.

FIG. 10 shows the effect of acute treatments with 2-hydroxyoleic acid(2OHOA) and its analogs 2-methyl oleic acid (2MOA), oleic acid (OA),palmitoleic acid (POA), cis-vaccenic acid (VA) and nervonic acid (NA).All the treatments carried out with 2-hydroxyoleic acid and the analogsthat comply with the general formula given above induced significantdecreases (*P<0.05, **P<0.001) in systolic arterial pressure inSprague-Dowley rats.

FIG. 11 shows the effect of 2-hydroxyoleic acid (OHOA) and its analogs,oleic acid (OA) and palmitoleic acid (POA), on body weight (3 dailyinjections of 30 mg/kg). The animals (Sprague-Dowley rats) had freeaccess to food and water at any time.

1. A method for treating a patient having a cancer selected from thegroup consisting of lung cancer and leukemia, the method comprisingadministering to the patient 2-hydroxyoleic acid in an amount effectiveto inhabit proliferation of the cancer.
 2. The method according to claim1, wherein the cancer is lung cancer.
 3. The method according to claim1, wherein the cancer is leukemia.
 4. A method for treating a patienthaving a brain metastasis formed from a lung adenocarcinoma, the methodcomprising administering to the patient 2-hydroxyoleic acid in an amounteffect proliferation of the brain metastasis.